Dr. Stanley has recently described a new disorder of hypoglycemia in infants with congenital hyperinsulinism and hyperammonemia (HI/HA syndrome). The principal investigator proposed a few years ago that this disorder is caused by mutations that result in excess activity of glutamate dehydrogenase (GDH). In the first 2 years of this grant, the PI has confirmed that these patients have dominant missense mutations of GDH that impair inhibitory responsiveness to its allosteric effector, GTP. A total of 52 mutations have been identified in GDH, in 3 different domains, which appear to be involved in inhibitory allosteric regulation of the enzyme. The PI has developed a method for expressing human GDH in bacteria to confirm that these mutations can produce defects in allosteric control. The goals of this application are to further define the molecular basis of HI/HA syndrome and to elucidate the consequences of GDH dysregulation on glucose and nitrogen metabolism. There are 3 specific aims: 1) to determine all of the locations of GDH mutations that can cause the HI/HA syndrome and correlate these mutation sites with differences in clinical phenotype; 2) to characterize, by expression of mutant GDH in bacteria, the effects of naturally occurring mutations and of additional designed mutations on enzyme activity and allosteric responsiveness; 3) to define mechanisms by which GDH mutations cause abnormal regulation of insulin secretion and ammonia detoxification using transgenic mice expressing mutant enzyme specifically in pancreatic beta-cells or in liver.